.t172 
Copy 1 



002 824 818 




BULLETIN OF THE 



No. 166 



Contribution from the Bureau of Animal Industry, A. D. Melvin, Chief 
January 22. 1915. 

(PROFESSIONAL PAPER.) 




OPHTHALMIC MALLEIN FOR THE DIAGNOSIS OF 

GLANDERS. 

By John R. Mohlek, Assistant Chief of Bureau, and Adolph ^ichhorn, Senior 
Bacteriologist, Pathological Division. 

INTRODUCTION. 

It is no longer doubted that in tlie work of controlling glanders 
the destruction of the infected animals should be given prompt con- 
sideration, and, if possible, the infection should be traced to its origm. 
Unfortunately, the nature of the disease is such that only a compara- 
tively small proportion of the cases can be recognized by the ordmary 
clinical examination, and as long as we limit our efforts to the destruc- 
tion of these cases the disease will contmue to spread. An effective 
c'ontrol can be accompUshed only by the eUmmation of all centers of 
infection of glanders. Therefore it is essential primarily to have a 
means of diagnosing accurately all forms of the disease. 

Numerous publications have been issued on the various methods 
of diagnosis, and it seems that while some favor a certain method or 
methods, others appear to produce sufficient evidence to point out 
the inadequacy of these methods. There is no question that m the 
last decade important progress has been made in the diagnosis of this 
disease. Smce the discovery of mallem, competent investigators have 
fruitfully studied this phase of the question of the control of glanders, 
and at the present tune we possess several methods by which we are 
reasonably sure of diagnosmg practically all cases of glanders. A 
minimum percentage of failures will probably always have to be con- 
tended with, as a good many factors enter into the execution of any 
test. 

In judging a method which would be the most satisfactory for the 
diagnosis of glanders various thmgs have to be taken into considera- 

NoTE.— This bulletin points out the advantageous and satisfactory use of the ophthalmic mallein test in 
the diagnosis of glanders and the necessity for prompt action on reactors to this test in eradicating this 
disease. Of interest to veterinarians and State !ive-stock,sanitary authorities. 
68247°-15 j^iJ':^"'^^- 



C^^-H 



3 



2 BULLETIN 1(J6, U. S. DEPARTMENT OF AGRICULTURE. Jm ' 

tion, but especially the reliability of the test. It should bo conven- 
ient, the results should be manifested as early as possible, the reaction 
should be distmct and well marked, and, probably the most important 
of all, it should be possible for the practicing veterinarian to apply 
the test. The last condition must be seriously considered, since the 
standing of the veterinarian m the community and the confidence of 
the pubhc in his work wordd be mor(> manifest if m suspected cases 
he could personally decide on the diagnosis instead of having to 
depend entirely on the results of serum tests made at some distant 
laboratory. 

VARIOUS METHODS FOR DIAGNOSING GLANDERS. 

It would require a great amount of space to enter into the liistory 
of the various methods of diagnosis and to enumerate the data we 
j)ossess on the different tests. The advantages and disadvantages of 
the various methods, es])ecially of the subcutaneous mallem tests, 
have been repeatedly published and are accessible to all those who 
are interested in the subject. There is no question that the sub- 
cutaneous mallem test is one of the valuable diagnostic agents for 
glanders, but no one can any longer deny that failures from this test 
are more numerous than are desu-able. As a matter of fact, the 
uncertainty of the results from this test caused numerous investi- 
gators to seek some other method which might replace the sub- 
cutaneous mallein test. Besides the failures resulting from it, the 
technic of executing the test, together with the time required for its 
determmation, make it unpopular with many veterinarians and sani- 
tary officers. 

Of the other tests which from time to time hav(> been devised for 
the diagnosis of glanders, the precipitation, the opsonic, and the con- 
glutination tests will not be considered, since the results obtained 
from them are not encouraging. 

For laboratory tests the combmed agglutination and complement- 
fixation test will no doubt remam the most satisfactory, and should 
always be applied in cases where doubt arises as to the results of 
other tests carried out by the practicmg veterinarian. These latter 
should be considered as accessory tests and provision should be made 
everywhere so that m case of doubt the serum could be subjected to 
the laboratory test mentioned, and the final decision should rest on 
its outcome. 

THE OPHTHALMIC MALLEIN TEST. 

During the last few years the ophthalmic mulU>iu test has gained 
great favor in the diagnosis of glanders. The popularity of the test 
is rapidly gauuiig wherever it has been applied, and among its sup- 
porters we find at the present time the greatest authorities on the 



d; op c 



OPHTHALMIC MALLEIN FOR THE DIAGNOSIS OF CLANDERS. 3 

subject of glanders and on clinical diagnosis. This method of testing 
is at present officially recognized in Austria, and tlie indications are 
that ere long it will constitute the official test in other countries. 
The results obtained m Austria, where the test has been employed 
for several years, are very gratifying, and Prof. Sclmui'er, of that 
coimtry, one of oui" greatest authorities on glanders, claims that the 
control of the disease can be very satisfactorily carried out by the 
application of the eye test, supplemented in doubtful cases by the 
agglutination test. Bavaria has recently adopted tliis method of 
diagnosis for official testmg. In Germany the method is also gaining 
m favor, and cuiTent veterinary literature contams expressions of 
satisfaction with this test from many German authorities. In the 
United States the Bureau of Animal Industry, m consideration of the 
favorable results obtained, has recognized this method of diagnosis 
for interstate shipments of equmes. The test has also been officially 
recognized by the Canadian authorities, and thus far no sanitary offi- 
cial connected with any of the States in this countiy has decMned to 
approve this test. 

The favorable results which have been obtained with this diagnostic 
method can no longer be denied. Its practicability is apparent, and 
its use in the control of glanders appears to be now an absolute 
necessity. 

— SIMPLICITY OF PROCEDURE. 

The ophthalmic test has a great advantage over others because of 
its very simple application. It may be readily executed by any 
veterinarian, and its other advantages are that the results are obtained 
in a comparatively short time and are, as a rule, distinct and defoiite. 
The simplicity of its application is plamly manifest when compared 
to the subcutaneous test, as it is only necessary to drop two to thi-ee 
drops of concentrated mallei n mto one of the eyes of the annual to 
be tested, or, by a still simpler procedure, to dij) a camel's-hair brush 
into mallem and introduce this mto the conjmictival sac of the anunal. 
The reaction usually commences in five to six hours after the intro- 
duction of the mallem and lasts from 24 to 30 hom's. A positive reac- 
tion is manifested by a purulent secretion from the tested eye. This 
may be very profuse or slight, somethnes associated with a severe 
conjunctivitis and edema of the lids, and at other times without any 
inflammatory symptoms bemg present. At times only a very small 
quantity of pus may be present in the iimer canthus of the eye. At 
other times the reaction may manifest a true pyorrhea. 

The reaction manifests itself ui varymg degrees in the animals, but 
the intensity of the reaction has no relation to the extent of the dis- 
ease in the reactor. 



4 BULLETIN Itilj, U. S. DEPAKTMEiS'T OF AGKICULTUflE. 

RELIABILITY OF THE TEST. 

The available data on the ophthaknic mallem test are suiRcient to 
draw conclusions as to the rehability of the method, and m Austria 
alone it has been applied on many thousands of cases with uniformly 
good results. 

In considering the good results obtained and the advantages of 
this method of testing, a concentrated mallein has been prepared for 
this purpose by the Bureau of Animal Industry, and this was made 
available to a number of practicing veterinarians who desired to give 
this method of testing a thorough trial. It has also been employed 
by inspectors of the Bureau of Animal Industry in their field work, 
and reports are accessible regarding its action for diagnostic pur- 
poses on more than 18,000 cases. The results from all sources were 
uniformly satisfactory. Practicing veterinarians who have given 
this method a trial have reported very favorably on the results, and 
the tests conducted by the bureau inspectors on several thousand 
animals were also satisfactory. The method has been applied here 
in Washington whenever possible, and recently in some immunizing 
tests of glanders conducted by the Bureau of Animal Industry there 
was a good opportunity to repeatedly employ this test. Jn all these 
instances the results were uniformly good. In cases of glanders there 
appeared a marked purulent conjunctivitis, and the reaction at times 
was so severe that the animal could not open its tested eye. 

BEST RESULTS WITH RAW MALLEIN. 

The essential factor in obtaining satisfactory results from the test 
appears to be in the use of the right kind of mallein. It must be by 
aU means a concentrated mallein, and apparently the best results 
follow the use of raw mallein, wliich, as a rule, represents the mallein 
obtained after the concentration of the filtrate from the bouiUon cul- 
tures of the glanders baciUi. The ordinary mallein used for subcu- 
taneous testing is not adaptable, and the failures wliich have been 
reported in the literature were without doubt, in the majority of 
cases, due to the fact that the mallein employed was not sufficiently 
concentrated. Marioth ^ correctly asserts that the reaction does not 
depend as much on the quality and quantity of the mallein as on its 
concentration. Our experiments in preserving such mallein with the 
ordinary quantity of 0.5 per cent carbolic acid showed that it does 
not interfere with the results of the test, although the lacrimation 
which follows immediately after the introduction of such mallein is 
more profuse than when carbolic acid has not been added, but this 
disappears within one or two hours after the application of the test. 

1 Monatsh. f. prakt. UerheUk., bd. 24, htt. 7/8, p. 340-373; htt. 9/10, p. 426-456. Stuttgart, 1913. 



OPHTHALMIC MALLEIN FOE THE DIAGNOSIS OF GLANDERS. 5 

PREPARATION OF THE MALLEIN. 

The concentrated mallein which has been. used for our work and 
which gave such satisfactory results was prepared at the request of 
the authors by and in cooperation with Mr. A. M. West, of the 
Biochemic Division, as follows: 

The media consists of bouillon containing 5 per cent glycerin, 1 per cent peptone, 
and 5 per cent NaCl. The reaction is that of the natural acidity of the meat, no acid 
or alkali being added. The flasks of media are inoculated with virulent cultures of 
Bacillus mallei and placed in the incubator at 37.5° C. for a period of two months or 
more. The stock cultures of B. mallei are kept on agar, and their \'irulence is re- 
newed when necessary by passage through a series of guinea pigs. 

The well-grown cultures show a heavy mass of organisms, which generally sinks to 
the bottom of the flask. This growth is of a whitish color splotched with brown. The 
cultures are then removed from the incubator and heated for one hour in the Arnold 
sterilizer. Then they are stored for two weeks in ai dark closet to settle. The clear 
liquid is then carefully decanted and the growth proper is discarded. 

A measured amount of the decanted licjuid is concentrated over a steam bath to 
one-third its volume. It is then filled into flasks and sterilized and again filtered 
while hot, first through one then through three paper filters. Next the clear liquid 
is passed through a Berkefeld filter. This is followed by a concentration to one-tenth 
its original volume and by sterilization. 

To the raw mallein, concentrated to one-tenth its original volume, is added 0.5 per 
cent carbolic acid and 20 per cent glycerin. Then the liquid is again concentrated 
to one-tenth its original volume, filtered while hot through filter paper, and sterilized. 
It is kept in a dark place for a week, and if upon inspection a precipitate is foimd the 
mallein is again passed through paper filters and sterilized. The finished product is 
a clear, sirupy, dark-brown liquid,' with a disagreeable odor. The mallein is then 
bottled, under aseptic conditions, in small ^•ials and is ready for use. 

It is advisable to provide the mallein for the tests in small vials, 
each containing about 1.5 c. c. of mallein, which is sufficient for testing 
15 horses. After the vial has been opened and part of the contents 
used for testing, especially if the mallein has been taken out with a 
camel's-hair brush, the remainder should not be used for tests applied 
on subsequent days, but should be discarded. 

THE USE OF DRY MALLEIN. 

Another form of mallein which has been used quite extensively for 
the eye test is the mallein siccum, or dry mallein. This represents an 
alcoholic precipitate of mallein. It is a fine gray powder and must 
be dissolved in water before it is used. The solution loses its effective- 
ness in a very short time and must be prepared fresh on the day of 
the test. Dr. K. F. Meyer, formerly of the University of Pennsyl- 
vania and now of the University of California, has used the dry 
maUein extensively, and at the present time this preparation is em- 
ployed in Pennsylvania for the application of the ophthalmic test. 
For this purpose two vials are sent out from the laboratories of the 
Pennsylvania Livestock Sanitary Board, one containing the pow- 



6 BULLETIN 166, U. 8. DEPARTMENT OF AGRICULTURE. 

ciered maUeiii and the other sterile or sahne water in quantities which 
will make a 5 per cent solution of mallein. The content of the bottle 
containing the fluid is poured into the bottle containing the mallein 
powder and the test solution is thus prepared. The results with this 
form of testing in Pennsylvania appear to be highly satisfactory, as 
may be seen from a publication by Dr. Meyer on the " Conjunctival 
reaction for glanders," in the May, 1913, mmiber of the Journal of 
Infectious Diseases. 

The advantages of the use of one as compared with the other of 
these forms of mallein for the eye test are not marked, as equally good ' 
results were obtained from the application of both forms of this 
])roduct. The fact that the preparation of the raw mallein is less 
laborious and expensive than the mallein siccum and that it is ready 
for use on opening the vial would probably give this product a greater 
popularity. It is only natural, however, that in the event subsequent 
extensive testings show the superiority of the dry mallein, it will be 
given preference over the raw product. 

METHOD OF APPLYING THE TEST. 

Before the application of the ophthalmic test the animals should be 
carefuUy examined to ascertain whether the eye shows conjunctivitis 
or other changes which are associated with suppuration. Should 
such be present the test should not be applied. 

The test consists in introducing into the conjunctival sac of the 
^ye several drops of either undiluted raw mallein or a solution of pre- 
cipitated mallein (0.1 to 0.2 c. c. per horse). This may be introduced 
either with the aid of a cameFs-hair brush or with an eyedropper. 
Only one eye is treated ; the other serves as a control for comparison 
of tlie reaction. For the testing of horses in the same stable the same 
dropper or camel's-hair brush may be used for all the animals. 

The results of the test should be recorded as follows: 

N=Negative — eye uuchauged. 
S=Suspicious — seromiicoue discharge. 
P-|-=Positive — seromucous discharge with purulent flakes. 
P4-+=Po8itive — distinct purulent discharge. 

P-f4- 4- = Positive — purulent discharge with swelling of the eyelids. 
P-f + + +=Positive — strong purulent discharge with swelling and gluing 
together of both lids. 



OPHTHALMIC MALLEIN FOR THE DIAGNOSIS OF GLANDERS. 7 

The following is a copy of Q. D. Form 69, Recortl of Ophthalmic 
Mallein Test, which is used by the Bureau of Animal Industry to 
record all official tests: 

[Obverse.] 
RECORD OF OPHTHALMIC MALLEIN TEST. 



No. 
of 
ani- 


Breed or 
markings. 


Sex 
(stal- 
lion, 
geld- 
ing, 
or 
mare). 


Age. 


Weiglit. 


Time 
of in- 
stilla- 
tion. 


Time of 
observation. 


Temperatures, 
if taken. 


Ante-mortem 
symptoms. 


Re- 
sults 

of 
test; 

de- 








(Give date and 
hour.) 


Be- 
fore. 


After. 


ci- 
sion. 









































1 

1 





















































































































































































































! 






































































































































































































1 























(Decision should be recorded in accordance with results obtained, by use of: [N]= Negative, eye un- 
changed. [S]= Suspicious, seromucous discharge. [P+]= Positive, seromucous discharge with purulent 
flakes. (P-t--{-]= Positive, distinct purulent discharge. [P +-!--(-]= Positive, purulent discharge with 
swelling of the eyelids. [P-l--f -l--f ]= Positive, strong purulent discharge with swelling and gluing together 
of the lids.) 

(Identify each animal by complete description; if necessary use two lines for an animal.) 



BULLETIN 166, U. S. DEPAETMENT OF AGRICULTURE. 

[Reverse.) 
RECORD OF REACTORS. 



No. of 


Disposition of reac- 
tors (slaughtered 
or quarantined). 


Date: 

Place of slaughter: . . . 




Post-mortem by: , 

































































































































(Q. D. Form 69.) 
U. S. Department of Agriculture, 

BUREAU OF ANIMAL INDUSTRY. 



Record of ophthalmic mallein test. 



Name of person making test 
Address: 

Dates: 

Owner: 

Address: 

Number passed: 

Number reacted: 

Number suspicious: 

Total number tested: 



EFFECT OF THE TEST ON GLANDERED AND HEALTHY ANIMALS. 

As soon as the mallein is introduced into the eye practically all 
animals show a lacrimation, increased reddening of the conjunctiva, 
and slight photophobia. No significance should be given to these 
symptoms. They disappear in one to two hours. 

Glandered animals are hy])ersensitive to mallein in a way that the 
administration of small quantities of mallein produces local inflam- 
matory processes. In larger quantities it produces a febrile general 
reaction. The hypersensitiveness appears as a rule during the third 
week after the infection, and reaches its height in the first few month.s 
after the infection. In the subsequent course it may subside in 
retrogressive cases even to the degree observed in healthy animals, 
but even in these cases various conditions may bring on an increased 
sensibility. 

The characteristic manifestations of the reaction for glanders 
commence as a rule in from 5 to 6 hours and last 24 to .36 hours, some- 



Bui. 1 66, U. S. Dept. of Agricultur 



Plate I. 





Fig. 1.— P + = Seromueous discharge with 
purulent flakes. 



Fig. 2.— P-f + = Distinct purulent discharge. 





Fig. 3.— P + + + = Purulent disehurge with 
swelling of the eyelids. 



Fig. 4.— P + + + + = Strong purulent dis- 
charge with .swelling and gluing together 
of both lids. 



Varying Degrees of Reactions in the Ophthalmic Mallein Test for Glanders. 



OPHTHALMIC MALLEIN FOE THE DIAGNOSIS OF GLANDERS. 9 

times longer. The reaction consists of a purulent discharge from 
the conjunctival sac which is typical, as well as swelling and gluing 
of the eyelids. It is advisable to examine the tested animals in a 
good light from 12 to 24 hours after the application of the test. 
Varymg degrees of reactions are illustrated in Plate I, figures 1 to 4. 

A suppurative discharge of varying quantities is considered a 
positive reaction. The conjunctiva and the eyeball should also be 
included in the examination after examining the discharge. A 
pseudo-reaction can be produced b}^ artificial or accidental irrita- 
tion of the eye. On the other hand the purulent discharge maj 
have been removed (either by the stable attendant or by the animals 
licking each other, etc.), and the positive result thus obliterated. 
In such cases dried pus may be frequently found on the ])arts around 
the eye. 

Generally the positive ophthalmic reactions are not accompanied 
by fever or systemic disturbances. Occasionally, however, affected 
horses are hypersensitive to such a degree that even the few drops 
of mallein placed in the eye may enter the circulation and produce 
fever. Therefore it is advisable, when possible, to accompany the 
ophthalmic reaction with temperature readings. For this purpose 
the temperature should be taken twice, the first time when the eye 
test is being made and the second time when it is judged. In a 
doubtful eye reaction, where there is an increased temperature of 
IJ degrees F., the test should be considered positive if the animal 
had a normal temperature at the time the test was made. As stated 
before, it should be remembered that the intensity of the reaction 
has no relation to the extent of the disease in the animal tested. 

In the absence of any secretion the test should be considered nega- 
tive. When there is a mucous secretion or lacrimation dui^g the 
period of reaction the test must be considered as atypical, and in 
such cases it may be repeated the same day, when, as a rule, the 
results are more confirming. 

The application of the ophthalmic test should not be repeated 
more than three times on the same animal within three months, as 
experiments show that the reaction after the third application within 
this short period usually loses its intensity in positive cases, and on 
subsequent tests may be entirely absent. In cases where the results 
of the second test immediately following the first test are atypical, 
the blood of such animal may be drawn and forwarded to a labora- 
tory for the serum diagnosis. From experience gained with the eye 
test such a procedure would become necessary only in a compara- 
tively few cases. In the control of glanders, animals may be retested 
every six months with satisfactory results. 



10 BULLETIN 166, U. S. DEPAETMENT OF AGRICULTURE. 

REPORT OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION ON 
THE OPHTHALMIC TEST. 

The special committee on the control of glanders of the American 
Veterinary Medical Association has issued a most excellent report 
on the various phases of diagnosis of glanders. The conclusions on 
the value of the eye test offered by this committee are in perfect 
accord with our findings, we therefore deem it advisable to include 
them in this paper, as follows: 

1. The ophthalmic test not only meets all the reqiiirements, but is without doubt 
the most convenient diagnostic method at our command. 

2. Its reliability compares favorably with any of the other tests available. 

3. The reaction is usually very distinct, and doubtful or atypical reactions are 
rather infrequent. 

4. The ophthalmic test has the advantage that it does not interfere with subsequent 
serum or other mallein tests if such are deemed necessary. 

5. The test may bP repeated within 24 hours on same or control eye. If another 
retest is necessarys it should not be made in less than three weeks. 

6. The ophthalmic test should be recognized by State and Federal authorities, 
since its reliability can no longer be doubted. 

7. In all atypical and doubtful cases of the ophthalmic test, the combined comple- 
ment-fixation and agglutination or subcutaneous mallein test should be utilized for 
confirmation. 8uch a procedure would minimize the failure and would assure the 
l)e8t results in the control of the disease in a single stable or in an entire community . 

CONCLUSION. 

The results achieved in Austria with the o})hthalmic test have 
been remarkably successful and deserve the most earnest considera- 
tion. The report of Prof. Schnurer on The Residts of the Diagnostic 
Procedure in Glanders in Austria is a convincing proof as to the 
value of the eye test in the control of glanders. The senior writer 
received a communication only a short time ago from Prof. Schnurer, 
and since it deals principally with the diagnostic value of the eye 
test, a quotation from the letter will no doubt be permissible: 

1 am at the present contemplating collecting the results of the eradication of glanders 
in Austria during the last three years (19I0-10T2). During this time 60,894 tests 
were undertaken on 47,973 horses. Of 272 cases which were found on post-mortem to 
be affected with glanders 240 (8S.2 per cent) were positive, 21 (7.7 per<'ent) gave an 
atypical reaction, while IJ (4 per cent) \yere negative. Of the 47,701 healthy horses, 
189 (0.39 per cent) were positive or atypical, the remaining 47.512 (99.61 per cent) 
gave a negative reaction. 

According to these results, therefore, the eradication of glanders is only a question 
of organization — that is, the malleinization of horses at the border and conscientious 
following up of all suspected horses. Such procedure would, without doubt, result 
in a complet<> eradication of glanders. At the Veterinary School of Austria we now 
have difficulty in showing the student cases of glanders, and for demonstration piu-- 
poses we are compelled to infect horses artificially, whereas several years ago we had 
every week at least one case of glanders in our clinics. 

I use as mallein at the present time a product which I, myself, prepare, which 
represents a bouillon filtrate from seven different strains of glanders bacilli which has 
becji concentrated to one-tenth of the original volume 



OPHTHALMIC MALLEIN FOR THE DIAGNOSIS OF GLANDERS. 11 

The Optimistic view of Prof. Schnurer is {'crttiinly justilied from 
the results he achieved, and clearly shows that with proper organiza- 
tion in the control work of glanders tte eradication of the disease is 
onU' a question of time. 

The eradication of outbreaks of glanders (^an not, of course, be 
altogether attributed to the eye test, since from the report of Never- 
mann, veterinar}' councilor of Prussia, glanders has diminished 
remarkably in that ct)initry, where they employ the combined 
complement-fixation and agglutination test for the diagnosis, while 
McGilvray has practically eradicated glanders from the Province of 
Manitoba by means of the subcutaneous mallein test. The method 
of testing by means of complement-fixation and agglutination is 
undoubtedly the most acciu-ate of any available, but since 'it can 
not be as conveniently applied as the eye test, its disadvantages are 
apparent. There is no doubt that with the application of either the 
eye test or the combined complement-fixation and agglutination 
tests, equally good results may be obtained provided that the work 
is conscientiously carried out and that all the reactors are destroyed 
without hesitation. 

As long as the authorities will limit themselves to the destruction 
of clinical cases only and will not take immediate action on reactors 
of the occult and latent character, glanders will not only continue 
to exist, but it will spread. 



WASHINGTON : i ;f ivp:r ^•^f KNT PitrNTixr; office : 1915 



